Chikungunya virus adaptation to Aedes albopictus mosquitoes does not correlate with acquisition of cholesterol dependence or decreased pH threshold for fusion reaction

Abstract

Background: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that recently caused several large scale outbreaks/epidemics of arthritic disease in tropics of Africa, Indian Ocean basin and South-East Asia. This re-emergence event was facilitated by genetic adaptation (E1-A226V substitution) of CHIKV to a newly significant mosquito vector for this virus; Aedes albopictus. However, the molecular mechanism explaining the positive effect of the E1-A226V mutation on CHIKV fitness in this vector remains largely unknown. Previously we demonstrated that the E1-A226V substitution is also associated with attenuated CHIKV growth in cells depleted by cholesterol.

Methods: In this study, using a panel of CHIKV clones that varies in sensitivity to cholesterol, we investigated the possible relationship between cholesterol dependence and Ae. albopictus infectivity.

Results: We demonstrated that there is no clear mechanistic correlation between these two phenotypes. We also showed that the E1-A226V mutation increases the pH dependence of the CHIKV fusion reaction; however, subsequent genetic analysis failed to support an association between CHIKV dependency on lower pH, and mosquito infectivity phenotypes.

Conclusion: the E1-A226V mutation probably acts at different steps of the CHIKV life cycle, affecting multiple functions of the virus.

Figure 1

Effect of S, T, G and I residues at E1-226 on growth of CHIKV in standard (A) and cholesterol-depleted (B) C6/36 cells. Standard cells were infected at an MOI of 1.0. Cholesterol-depleted cells were infected at an MOI of 0.1. Viral titers are expressed as Log₁₀TCID₅₀/mL ± standard deviation of 2 independent experiments. hpi - hours post-infection.

Figure 2

Effect of S, T, G and I residues at E1-226 on CHIKV infectivity for cholesterol-depleted C6/36 cells. Standard (blue bars) and cholesterol-depleted (yellow bars) C6/36 cells were infected with serial dilutions of eGFP expressing CHIKV containing indicated residues at E1-226. Results are normalized for 10⁶ viral infections of standard C6/36 cells. Data indicate an average of three experiments ± standard deviation. IC-infectious center.

Figure 3

Effect of P, F, M, H and L residues at E1-226 on growth of CHIKV in standard (A) and cholesterol-depleted (B) C6/36 cells. Standard cells were infected at an MOI of 1.0. Cholesterol-depleted cells were infected at an MOI of 0.1. Viral titers are expressed as Log₁₀TCID₅₀/mL ± standard deviation of 2 independent experiments.

Figure 4

Effect of P, F, M and H residues at E1-226 on CHIKV infectivity for cholesterol-depleted C6/36 cells. Standard (blue bars) and cholesterol-depleted (yellow bars) C6/36 cells were infected with serial dilutions of eGFP expressing CHIKV containing indicated residues at E1-226. Results are normalized for 10⁶ viral infections of standard C6/36 cells. Data indicate an average of three experiments ± standard deviation.

Figure 5

Growth kinetics of CHIKV with mutations in E1 protein identified in Clone#1 virus in standard (A) and cholesterol-depleted (B) C6/36 cells. Standard cells were infected at an MOI of 1.0. Cholesterol-depleted cells were infected at an MOI of 0.1. Viral titers are expressed as Log₁₀TCID₅₀/mL ± standard deviation of 2 independent experiments.

Figure 6

Effect of NH₄Cl on competition between LR-ApaI-226V and LR-226A viruses for growth in C6/36 (A) and BHK-21 cells (B). Thirty minutes prior to infection cells were pre-incubated with L-15 media containing various amounts of NH₄Cl. Then cells were infected with 1:1 mixture of LR-ApaI-226V and LR-226A viruses (10⁷pfu/mL) and incubated for 48 h in L-15 media containing various amounts of NH₄Cl. Cell culture supernatants were harvested and used for RNA extraction or titration on Vero cells. Virus RNA was processed as discussed previously [15].

Figure 7

Effect of E1-A226V mutation on pH dependence of CHIKV induced cell-cell fusion. C6/36 cells infected with LR-GFP-226A or LR-GFP-226V viruses were incubated for 2 min with L-15 medium whose pH was previously adjusted to desired values. The reaction was abrogated by replacement of the fusion medium with 0.5 mL of standard L-15 medium. Percent of fusion was calculated as (1-c/n)x100%, where c is a number of eGFP expressing cells, n is number of nuclei (n≥70). Two independent experiments were performed for each virus (i and ii). Each individual experiment was performed in duplicates and results are expressed as an average of duplicates.

Figure 8

Effect of mutations at E1-226 (A) and [E1-66/E1-70] (B) on pH dependence of CHIKV induced cell-cell fusion. C6/36 cells were infected with eGFP expressing viruses and processed as described in legend for Figure 7. Experiments were performed in duplicates for each virus, and results are expressed as an average of duplicates.